ETAA1 promotes genome stability through activation of ATR

Abstract

The ATR kinase controls cell cycle transitions and the DNA damage response. Budding yeast contain three activators of Mec1ATR; however, only TOPBP1 is known to activate ATR in vertebrates. We identified ETAA1 as a replication stress response protein in two proteomic screens. ETAA1-deficient cells accumulate double-strand breaks and other hallmarks of genome instability and are also hypersensitive to replication stress. ETAA1 contains two RPA-interaction motifs that localize ETAA1 to stalled replication forks. It binds ATR/ATRIP directly using a motif with sequence similarity to the TOPBP1 ATR-activation domain; and like TOPBP1, ETAA1 acts as a direct ATR activator. ETAA1 functions in parallel to TOPBP1 to regulate ATR and maintain genome stability. To examine how each activator contributes to ATR signaling, we used quantitative phosphoproteomics to identify changes in protein phosphorylation in ETAA1- or TOPBP1-deficient cells. Gene ontology analysis of TOPBP1 and ETAA1-dependent phosphoproteins revealed TOPBP1 to be a primary ATR activator for replication stress while ETAA1 regulates mitotic ATR signaling at centromeres. Inactivation of ATR or ETAA1 results in decreased Aurora B kinase activity during mitosis. Additionally, ATR activation by ETAA1 is required for proper chromosome attachment during metaphase and for a fully functional spindle assembly checkpoint response. Thus, we conclude that ETAA1 and TOPBP1 regulate distinct aspects of ATR signaling through activation of ATR.